Proteins are the major components of cells. The spatiotemporal expression pattern and the subcellular localization of proteins determines the shape, structure, and function of cells. Proteins are assembled from 20, different amino acids, each with a distinct side chain and chemical property. This provides for enormous variety in the chemical properties of proteins and the activities they exhibit.
In addition, many proteins are dynamically regulated such that their activity is altered in response to certain intrinsic or extrinsic cues. One form of regulation involves covalent modification of the regulated protein. An example of such a covalent modification is the substitution of a phosphate group for a hydroxyl group in the side chain of an amino acid (phosphorylation). Such modification is often associated with an alteration in the activity of a protein. Protein kinases can recognize specific protein substrates and catalyze the phosphorylation of serine, threonine, or tyrosine residues on their protein substrates. Such substrate proteins capable of being phosphorylated are referred to as phosphoproteins. Once phosphorylated, a substrate protein may have its phosphorylated residue converted back to a hydroxylated one by the action of a protein phosphatase which specifically recognizes the substrate protein. Protein phosphatases catalyze the replacement of phosphate groups by hydroxyl groups on serine, threonine, or tyrosine residues. Through the action of kinases and phosphatases a protein may be reversibly phosphorylated on a multiplicity of residues and its activity may be regulated thereby.
Many kinases and phosphatases are known and play important roles in signal transduction. Many kinases and phosphatases are phosphoproteins, the kinase and phosphatase activities of which are regulated by phosphorylation. The phosphorylation of kinases and phosphatases is often regulated by other or same type kinases and phosphatases. In this way, the regulators of signal transduction are themselves regulated as part of a signaling cascade.
Many intracellular kinases and phosphatases transduce signals in response to extracellular signals that stimulate receptors at the cell surface. As a result, extracellular signals induce changes in the phosphorylation and activation states of many proteins in receptive cells. For example, many mitogens induce JNK and MAPK signaling activity which causes cell to proliferate. As another example, neurotrophic factors have been shown to induce AKT signaling activity which supports neuronal survival. In addition, many diseases, such as cancer, are associated with an alteration in the activation state of many signaling proteins. In many instances, the signaling activity observed in cancer cells is reminiscent of growth factor stimulated signaling activity in non-cancer cells.
Some lipids of the cell membrane are also substrates for kinases. For example, phosphatidylinositol 3-kinase (PI3K) specifically recognizes phosphatidylinositol 4,5-diphosphate (PIP2) and catalyzes its phosphorylation at the 3′ position of the inositol moiety. PIP2, is generated by the activity of another kinase, phosphatidylinositol 4-kinase which catalyzes phosphorylation of phosphatidylinositol at the 4′ and 5′ positions of the inositol moiety. PI3K regulates a variety of cellular functions and is activated by growth factors such as nerve growth factor (NGF), platelet derived growth factor (PDGF), insulin receptor substrate 1, (IRS-1) and CD28. Many of the proteins that activate PI3K also activate GTPases, predominantly the ras family, which in turn activate PI3K by binding to its p110, subunit. The direct pathway from tyrosine phosphorylated receptors to PI3K and activation of PI3K by GTPase proteins function synergistically. PI3K is a heterodimeric enzyme composed of a catalytic subunit (p110) and a regulatory subunit. Several genes encoding regulatory subunits of PI3K have been identified, including p85α and p85β. In addition, a number of splice variants of p85α are known (including p55α and p50α).
It is understood that kinase signaling cascades play an important role in nearly every critical decision process in cells (for review see Hunter, Cell 100:113-127, 2000). Determining the role of specific signal transduction pathways in a given system has been aided by the advent of pharmacological inhibitors for specific kinases. However, monitoring activities for such kinases, for example, protein kinase B/AKT, cJun-N-terminal kinase (JNK), p38, mitogen activated protein kinase (p38), p44/42, ERK1/2, (ERK), PKA, PKC or TYK2, or others, is usually dependent upon in vitro kinase assays or more recently by immunoblotting to determine the phosphorylation state of these proteins using phospho-specific antibodies. To date it has not been possible to correlate rare subpopulations of cells with the activation state of kinases in important signaling pathways.
Another form of protein regulation involves proteolytic cleavage of a peptide bond. While random or misdirected proteolytic cleavage may be detrimental to the activity of a protein, many proteins are activated by the action of proteases that recognize and cleave specific peptide bonds. Many proteins derive from precursor proteins, or pro-proteins, which give rise to a mature isoform of the protein following proteolytic cleavage of specific peptide bonds. Many growth factors are synthesized and processed in this manner, with a mature isoform of the protein typically possessing a biological activity not exhibited by the precursor form. Many enzymes are also synthesized and processed in this manner, with a mature isoform of the protein typically being enzymatically active, and the precursor form of the protein being enzymatically inactive. This type of regulation is apparently not reversible. Thus, to inhibit the activity of a proteolytically activated protein, mechanisms other than de-cleavage must be used. It is the case that many proteolytically activated proteins are relatively short lived proteins.
Among the enzymes that are proteolytically activated are the caspases. The caspases are an important class of proteases that mediate programmed cell death (referred to in the art as “apoptosis”). Caspases are constitutively present in most cells, residing in the cytosol as a single chain proenzyme. These are activated to fully functional proteases by a first proteolytic cleavage to divide the chain into large and small caspase subunits and a second cleavage to remove the N-terminal domain. The subunits assemble into a tetramer with two active sites (Green, Cell 94:695-698, 1998).
Antibodies are particularly useful for the study of proteins. Antibodies are a large family of glycoproteins that specifically bind antigens, and do so with a high affinity. Antibodies comprise a constant domain and a variable domain. The variable domain of an antibody binds to antigen. Polyclonal antibodies comprise a multiplicity of variable region types. Monoclonal antibodies comprise a single type of variable region, and are thus more likely to specifically bind to fewer proteins. Monoclonal and polyclonal antibodies are routinely generated by means known in the art, and have been raised against many known proteins for use in both research and therapy. Antibodies may be used to determine the presence of antigens or proteins to which they specifically bind. Well known immunochemical methods that employ this approach include Western blotting, immunoprecipitation, and enzyme linked immunoassay (ELISA).
In particular, antibodies may be used to determine the presence of a specific isoform of a protein, especially, when the isoform possesses an antigen not present in other isoforms of the protein, or when the isoform lacks an antigen not present in other isoforms of the protein. Thus, the isoform can be antigenically distinct from other isoforms of the same protein. The antigenically distinct isoform may uniquely possess or lack a covalently attached moiety, or may be in a conformation distinct from other isoforms and thereby present the same amino acid sequence in an antigenically distinguishable way.
For example, the determination of PI3K activity has typically involved immunoprecipitation of PI3K and an in vitro enzymatic assay using phospholipids and labeled ATP. Recently, antibodies that specifically bind to phosphatidylinositol 4,5-diphosphate and phosphatidylinositol 3,4,5-triphosphate have been developed (Molecular Probes, A-21327, anti-phosphatidylinositol 4,5-diphosphate, mouse IgM monoclonal 2C11; A-21328, anti-phosphatidylinositol 3,4,5-triphosphate, mouse IgM monoclonal RC6F8). These antibodies serve as useful tools for the detection of PI3K substrate (PIP2) and product (PIP3), and can be used to determine the activity of PI3K.
Biochemical investigations of protein expression and function have traditionally focused on one or a few proteins at a time. This has largely been due to limitations imposed by the available investigative techniques. It is well understood that a cell's normal and abnormal physiology is the product of a large number of proteins participating in a large number of molecular interactions. For the purposes of designing treatments and cures, an understanding of the mechanisms underlying disease is desired. Such an understanding requires consideration of multiple proteins and their molecular interactions over time. In this regard, one important experimental hurdle is how to analyze large numbers of proteins in a single experiment, for example to determine a protein expression profile and, more specifically, active protein profiling for a single protein sample, or even for a single cell.
In addition to qualitative measurements of protein expression in cells, quantitative measures of protein expression are desirable. It is well known that many proteins can function differently at different expression levels, and such differences in protein function can lead to differences in cell physiology.
It is highly desirable to determine expression levels of particular isoforms of proteins in a sample, given that different isoforms of a protein can have different activities. For example, proteins involved in signal transduction can often exist in two or more forms, and it is usually a particular form of the signaling protein that transmits a signal that mediates the effects of a signaling pathway. Conversion from one form to another is often used to turn a signal on or off through a change in protein activity.
In addition, in order to study protein expression and function, it is often necessary to immobilize proteins on a solid support. In Western blot analysis, proteins of interest are first separated by electrophoresis and then transferred and immobilized onto a nitrocellulose or a polyvinylidene difluoride (PVDF) membrane. In the phage display screening of a protein expression library, several hundred thousand proteins expressed by phages are immobilized on membranes. In both Western blotting and phage display screening, proteins are immobilized non-covalently. The protein of interest is then selected by its unique property, i.e., interaction with an antibody. In some other applications such as immunoprecipitation and affinity purification, agents (e.g., antibodies, ligands) are covalently conjugated onto solid supports (e.g., agarose beads) through their primary amines, sulfhydryls or other reactive groups. In general, proteins retain their abilities of interacting with other proteins or ligands after immobilization. However, even with the immobilization of a multiplicity of proteins from a sample, the problems of simultaneous detection of protein expression, protein form, and protein level for a multiplicity of proteins remains.
Thus, an object of the present invention is to overcome the problems described above. Accordingly, the present invention provides an approach for the simultaneous determination of the activation states of a plurality of proteins in single cells. This approach permits the rapid detection of heterogeneity in a complex cell population based on protein activation states, and the identification of cellular subsets that exhibit correlated changes in protein activation within the cell population. Moreover, this approach allows the correlation of cellular activities or properties, such as surface molecule expression or cell granularity, with protein activation at the single cell level.